Antidiarrheal activity of Clitorea ternatea Linn. (Fabaceae) ethanol leaf extract in rats.

BACKGROUND: Clitorea ternatea Linn. (Fabaceae) is a plant which is traditionally used for the treatment of wide range of pharmacological activities including antimicrobial, antipyretic, anti-inflammatory, analgesic, diuretic, local anesthetic, antidiabetic, insecticidal, blood platelet aggregation-inhibiting and diarrhea in India. However, scientific evidence does not exist in any literature to corroborate the claim of therapeutic success of the plant species in diarrhea. AIM OF THE STUDY: The core aim of the present study is to evaluate the antidiarrheal activity of C. ternatea ethanol extract (CTE).
MATERIALS AND METHODS: The antidiarrheal activity was evaluated using castor oil and magnesium sulfate (MgSO4)-induced diarrhea method. The effects of CTE on gastrointestinal motility, intestinal transit and enteropooling were also examined in rats.
RESULTS: CTE (100-400 mg/kg, p.o.) produced dose-dependent and significant (P < 0.05-0.01) protection of rats against castor oil and MgSO4-induced diarrhea, inhibited intestinal transit and delayed gastric emptying. CTE dose dependently and significantly delayed the onset of castor oil and MgSO4-induced diarrhea, decreased the frequency of defecation and reduced the severity of diarrhea in the rats compared with loperamide (10 mg/kg, p.o.).
CONCLUSION: These findings confirm the ethno medicinal use of C. ternatea as a valuable natural remedy for the treatment, management and/or control of diarrhea.

Introduction

Since ancient times, diarrhea has been recognized as one of the most important health problems afflicting humanity, particularly those populations in socioeconomically backward, developing and third world countries. Globally, diarrhea has been estimated to kill about 2.2 million people annually, the majority of whom are infants and children below the age of 5 years.[1]

Diarrhea is a disorder characterized by discharge of semisolid or watery fecal matter from the bowel three or more times in a day.[23] It involves an increase in the fluidity, volume and frequency of bowel movements, increased frequency of bowel sound, wet stools and abdominal pain, accompanied by increased secretion and decreased absorption of fluid and thus loss of water and electrolytes.[45]

In general, the treatment of diarrhea is nonspecific and is usually aimed at reducing the discomfort and inconvenience of frequent bowel movements.[6] To overcome the menace of diarrhea in developing countries, especially the discomfort and inconvenience of frequent bowel movements, the World Health Organization has introduced a program for diarrheal control which involves the use of traditional herbal medicines. Several African medicinal plants have been reported to be useful in the treatment, management and/or control of diarrhea.[78910]

Clitorea ternatea L. (Family: Fabaceae) is a perennial twing herb, stems are terete, more or less pubescent. The roots have a sharp bitter taste and are useful in severe bronchitis, asthma, inflammation, diarrhea and fever.[1112] The fatty acid content of C. ternatea seeds includes palmitic, stearic, oleic, linoleic and linolenic acids.[13] The seeds also contain beta-sitosterol.[14] C. ternatea possesses sedative action.[15] It enhances memory[16] and increases acetylcholine content in rats.[17] Its nootropic, anxiolytic, antidepressant, anticonvulsant,[18] antipyretic, anti-inflammatory and analgesic activities,[19] antidiabetic,[20] antiasthamatic[21] and hepatoprotective[22] properties are also reported. Since a systematic study of its antidiarrheal activity has not been undertaken, this study was done to evaluate the antidiarrheal activity of C. ternatea ethanol extract (CTE) in rats.

Materials and Methods

Ethical considerations

Experimental procedures and protocols used in this study were approved by the Institutional animal ethics committee (MES/COP/IAEC/01/2010-11) and the care of the laboratory animals was taken as per the current committee for the purpose of control and supervision of experiments on animals regulations.

Plant materials

Fresh leaves of C. ternatea L. (Fabaceae) were collected in September 2010 from the Mahatma Phule Krishi Vidyapeeth, Rahuri, Maharashtra, India and were authenticated by Dr. J. Jayanti, Botanist, Botanical Survey of India (BSI), Pune. A voucher specimen (BSI/WRC/Tech./2011/581) has been kept in herbarium, BSI for future reference.

Preparation of plant extract

Fresh leaves of C. ternatea L. were washed with distilled water, shed dried and later powdered. This powder was then defatted with petroleum ether which was further macerated with ethyl alcohol for 72 h with occasional shaking. It was then filtered and evaporated. The yield of CTE was 3.6% w/w. Purified aliquot portions of the crude plant extract were weighed and dissolved in distilled water (at room temperature) for use on each day of the experiments.

Animals

Healthy adult, wistar rats (Rattus norvegicus) of both (male/female) sexes weighing 150–200 g were obtained from the animal house. All the animals were housed in clean polypropylene cages placed in well-ventilated house conditions of temperature and humidity with a 12 h light/dark cycle. They were allowed free access to food (standard pellet diet) and drinking tap water ad libitum, except when fasting was required during the study. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care.

Acute toxicity testing

The present study was conducted according to the organization for economic cooperation and development (OECD) revised fixed-dose procedure for acute toxicity testing (OECD guideline 420, 2001). Two groups of five healthy female albino wistar rats (3 months old, 150–200 g b. wt. were administered a limit dose of 2000 and 5000 mg/kg of the CTE and animals were observed for mortality and clinical signs for the 1st hour, then hourly for 3 h and finally periodically until 48 h. All of the experimental animals were maintained under close observation for 14 days and the number of rats that died within the study period were noted. The lethal dose (LD50) was predicted to be above 2000 or 5000 mg/kg if three or more rats survived.[23]

Evaluation of antidiarrheal activity of Clitorea ternatea ethanol extract

Castor oil-induced diarrhea in rats

Wistar albino rats were divided into five groups of six animals (n = 6) each. All rats were fasted for 18 h and received castor oil at a dose of 1 ml/animal orally (p.o.) using orogastric tubes for induction of diarrhea.[24] Thirty minutes after castor oil administration, rats of group I (control) received 1 ml/100 g of 0.9% NaCl in distilled water (normal saline), group II (reference) received standard drug, loperamide (3 mg/kg p.o.) and rats of groups III, IV and V received 100, 200 and 400 mg/kg CTE, p.o., respectively. The animals were placed separately in metabolic cages over white clean Whatman filter paper, which was changed every hour. The severity of diarrhea was assessed each hour for 4 h. The total number of diarrhea feces of the control group was considered 100%.

Magnesium sulfate-induced diarrhea

Wistar albino rats were divided into five groups of six animals (n = 6) each. All rats were fasted for 18 h and received magnesium sulfate (MgSO4) at a dose of 2 g/kg orally (p.o.) using orogastric tubes for induction of diarrhea.[25] Thirty minutes after MgSO4 administration, rats of group I (control) received 1 ml/100 g of 0.9% NaCl in distilled water (normal saline), group II (reference) received standard drug, loperamide (3 mg/kg p.o.) and rats of groups III, IV and V received 100, 200 and 400 mg/kg CTE, p.o., respectively. The animals were placed separately in metabolic cages over white clean Whatman filter paper, which was changed every hour. The severity of diarrhea was assessed each hour for 4 h. The total number of diarrheal feces of the control group was considered as 100%.

% Inhibition of diarrhea = (total weight of feces of control group − total weight of feces of test group) × 100/total weight of feces of control group

Measurement of gastrointestinal transit time using charcoal

Wistar albino rats were fasted for 18 h and divided into five groups of six animals each. Castor oil (1 ml) was administered orally to the animals. One hour later, group I (control) was administered 1 ml/100 g of 0.9% NaCl in distilled water (normal saline), group II (reference) received standard drug, atropine sulfate (5 mg/kg p.o.) and rats of groups III, IV, and V received 100, 200 and 400 mg/kg CTE p.o., respectively. After 30 min of the administration, 1 ml of charcoal meal (10% suspension in 5% gum acacia) as a marker diet was given orally to rats in all groups and immediately rats were sacrificed by ether (20% v/v) anesthesia and small intestine was separated from mesentery avoiding being stretched. For each animal, gastrointestinal (GI) transit was calculated as percentage distance travelled by charcoal meal to the total length of intestine. The inhibitory effect of CTE on GI transit was calculated relative to atropine sulfate.[26]

Measurement of castor oil-induced intestinal enteropooling and fluid accumulation

Castor oil-induced enteropooling was determined by the method of Robert et al.[27] Wistar albino rats were fasted for 18 h and divided into five groups of six animals each. Castor oil (1 ml) was administered orally to the animals. One hour later, group I (control) was administered 1 ml/100 g of 0.9% NaCl in distilled water (normal saline), group II (reference) received standard drug, atropine sulfate (5 mg/kg p.o.) and rats of groups III, IV and V received 100, 200 and 400 mg/kg CTE, p.o., respectively. After 2 h of treatment, the rats were sacrificed by ether anesthesia. The edges of the intestine from pylorus to cecum were tied with thread and the intestine was removed and weighed. Intestinal content was collected by milking into a graduated tube and the volume was measured. The intestine was reweighed and the differences between full and empty intestines were calculated.

Statistical analysis

The results were expressed as mean ± standard error of mean; the level of statistical significance for the observed difference in the mean P < 0.05 was considered as statistically significant. Statistical analysis was done using one-way analysis of variance followed by Dunnette’s multiple comparison test.

Results

Preliminary phytochemical screening

The preliminary phytochemical study showed the presence of steroids, terpenoids, alkaloids, tannins, phenolic compounds, flavonoids, sugars and amino acids.

Acute toxicity study

Oral administration of CTE was found safe at a dose of 2000 mg/kg, p.o. and produced no signs of toxicity. However, at 5 g/kg, CTE caused slow movement of animal, decreased aggressiveness and altered touch and pain sensibility, but did not caused any negative behavioral changes such as excitement, respiratory distress, convulsions or coma. No mortality was observed up to 14 days. Hence, the median LD50 of the CTE was then >2000 mg/kg body weight. Therefore, doses 100, 200 and 400 mg/kg birth weight were selected for all in vivo experiments.

Castor oil-induced diarrhea

The CTE was found to be effective against castor oil-induced diarrhea in rats at doses of 100, 200 and 400 mg/kg body weight as compared to control reflected by slower onset of diarrhea, reduced frequency of diarrhea and number of wet stools. Further, at the end, there was a significant 79.25%, 20.87%, 41.75% and 70.87% reduction of diarrheal feces with the loperamide and CTE (100, 200 and 400 mg/kg) respectively in a dose-dependent manner when compared with control group [Table 1].

Table 1

Effect of Clitorea ternatea leaf ethanol extract on castor oil-induced diarrhea in rats

Magnesium sulfate-induced diarrhea

The CTE was found to be effective against MgSO4 induced diarrhea in rats at doses of 100, 200 and 400 mg/kg body weight as compared to control reflected by slower onset of diarrhea, reduced frequency of diarrhea and number of wet stools with a significant 69.58%, 47.78%, 56.52% and 65.27% reduction of diarrheal feces with the loperamide and CTE (100, 200 and 400 mg/kg), respectively, in a dose-dependent manner when compared with control group [Table 2].

Table 2

Effect of Clitorea ternatea leaf ethanol extract on magnesium sulfate-induced diarrhea in rats

Measurement of gastrointestinal transit using charcoal meal

Administration of CTE shows significant reduction of GI transit against castor oil-induced diarrhea in rats with a significant 43.52%, 12.04%, 18.05% and 26.86% reduction of GI transit with atropine and CTE (100, 200 and 400 mg/kg), respectively in a dose-dependent manner when compared with control [Table 3].

Table 3

Effect of Clitorea ternatea leaf ethanol extract on castor oil-induced intestinal propulsion in rats (charcoal meal study)

Measurement of castor oil-induced intestinal enteropooling and fluid accumulation

Administration of CTE was found to possess an antienteropooling properties in castor oil-induced experimental animals with a significant inhibition of intestinal content by 84.42%, 43.71%, 59.38% and 71.85% from atropine and CTE (100, 200 and 400 mg/kg), respectively, in a dose-dependent manner when compared with control [Table 4].

Table 4

Effect of Clitorea ternatea leaf ethanol extract on castor oil-induced intestinal enteropooling and fluid accumulation in rats

Discussion

Diarrhea is the frequent passage of liquid feces and it involves both an increase in the motility of the GI tract along with increased secretion and decreased absorption of fluid and thus a loss of electrolytes (particularly sodium) and water.[28] Therefore, to restore personal comfort and convenience, many patients require antidiarrheal therapy and treatment is carried out to achieve other objectives such as increased resistance to flow (segmental contraction, decreased propulsion and peristalsis) and increased mucosal absorption or decreased secretion.[2930] The present investigation involves evaluation of the antidiarrheal potential of C. ternatea against castor oil and MgSO4-induced diarrhea. CTE effect was also investigated on GI transit using charcoal meal in castor oil-induced diarrheal rats in comparison to concerning actions of drugs such as atropine sulfate in reducing GI transit.

Castor oil, a very effective laxative is hydrolyzed in the upper small intestine to ricinoleic acid,[31] which can stimulate fluid secretion, inhibit water and electrolyte absorption, reduce active Na+ and K+ absorption and decrease Na+, K+-ATPase in the small intestine and colon.[2432] Castor oil also increases the peristaltic activity and produces permeability changes in the intestinal mucosal membrane to electrolytes and water. Furthermore, ricinoleic acid can also lead to the release of endogenous prostaglandins,[33] which play an important role in the modulation of function of GI tract and stimulate motility and secretion leading to diarrhea.[34] In this study, the results showed that CTE reduced castor oil-induced diarrhea as well as the number of diarrheal feces and total weight of feces in a dose-dependent manner, which could be taken as antidiarrheal potential. Loperamide is one of the most efficacious and widely employed antidiarrheal drugs which effectively antagonized the diarrhea induced by castor oil[35] and prostaglandiner cholera toxin.[36] The therapeutic effect of loperamide is believed to be due to its antimotility and antisecretory activity.[37]

MgSO4 has been reported to induce diarrhea by increasing the volume of intestinal content through prevention of reabsorption of water. It has been demonstrated that it promotes the release of cholecystokinin from the duodenal mucosa, which increases the secretion and motility of small intestine and thereby prevents the reabsorption of sodium, chloride and water.[38] The CTE was also found to reduce MgSO4-induced diarrhea significantly which could be due to increased absorption of water and electrolytes.

The CTE exhibited a significant antidiarrheal effect on GI transit using charcoal meal in rats. Hypermotility characterizes forms of diarrhea where the secreting component is not the causative factor. The CTE suppressed the propulsive movement or GI transit of charcoal meal, which clearly indicates that extract, may be capable of reducing the frequency of stools in diarrheal conditions. Anticholinergic agents are known to inhibit GI hyper-motility. Castor oil-induced GI hyper-motility has been suggested to be indirectly mediated by cholinergic system since it is inhibited by atropine sulfate, a known anticholinergic agent.[39] CTE was found to possess an antienteropooling in castor oil-induced diarrheal rats by reducing both weight and volume of intestinal content. These effects are direct consequences of reduced water and electrolytes secretion in small intestine, suggesting that extract may enhance water and electrolyte absorption from intestinal lumen. Phytochemical screening revealed the presence of numerous constituents such as steroids, terpenoids, alkaloids, tannins, phenolic compounds, flavonoids, sugars and amino acids. Antidiarrheal properties of medicinal plants were found to be due to tannins, flavonoids, alkaloids, saponins, reducing sugar, sterol and terpenes.[40414243] Hence, tannins, reducing sugars and sterols may be responsible for antidiarrheal potential of CTE.

Conclusion

C. ternatea ethanol extract (CTE) produced dose-dependent and significant (P < 0.05–0.01) protection of rats against castor oil and MgSO4-induced diarrhea, inhibited intestinal transit and delayed gastric emptying and inhibited castor oil-induced enteropooling in rats when compared with standard drugs such as loperamide and atropine sulfate, respectively. These findings demonstrate that C. ternatea has the potential to treat GI disorders such as diarrhea owing to its antidiarrheal effect. Further studies are necessary to substantiate above claim and to work out exact mechanism of action involved in antidiarrheal activity of this plant.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.